The invention relates to a microfluidic device of an electrophoretic apparatus for analyzing a small quantity of a sample at a high speed with a high resolution, and an electrophoretic analyzing method using the same.
An electrophoretic analysis using a microfluidic device has been widely used in a field of biochemistry, molecular biology, clinical medical, and analysis of deoxyribonucleic acids (hereinafter simply referred to “DNA”) and proteins.
When a small quantity of DNA, protein or the like is analyzed, an electrophoresis has been conventionally used. As an example of a device, there has been known a capillary electrophoretic apparatus. In the capillary electrophoretic apparatus, a migration buffer is filled in a glass capillary (hereinafter referred to simply “capillary”) having an inner diameter of less than 100 μm. After a sample is injected into one end of the capillary, a high voltage is applied between both ends thereof so that a substance to be analyzed travels in the capillary. The interior of the capillary has a large surface area relative to a capacity, i.e. a high cooling effect. Therefore, it is possible to apply a high voltage, so that a small quantity of a sample such as DNA can be analyzed at a high speed with a high resolution.
Recently, instead of the capillary whose handling is troublesome, as a device with a high analyzing speed and a small size, there has been proposed a microfluidic device, i.e. electrophoretic chip device, formed of joined two base plates, as disclosed in D. J. Harrison et al., Science 261 (1993) pp. 895–897, and Anal. Chem. Acta 283 (1993) pp. 361–366.
An example of the microfluidic device is shown in FIGS. 4(A)–4(C). The microfluidic device is formed of a pair of transparent base plates 1 and 2 generally made of glass, quartz, resin or the like. A loading channel 4 and a separating channel 5, which cross each other, are formed in one base member 2, as shown in FIG. 4(B). Reservoirs 3 are formed in the other base member 1 as a through-hole at positions corresponding to respective both ends of the loading channel 4 and the separation channel 5, as shown in FIG. 4(A). The base plates 1 and 2 are laminated and joined together, as shown in FIG. 4(C). In the conventional microfluidic device described above, the sample is basically injected into the channel with a cross injector design formed of the loading channel 4 and the separating channel 5 crossing each other.
When such a microfluidic device is used, a migration buffer is injected into the loading channel 4 and the separation channel 5 from any one of the reservoirs 3. Then, about 1–2 μl (micro liter) of a sample is injected into the reservoir 3 located at one end of the loading channel 4. A predetermined voltage is applied to plural portions by pinching or the like through electrodes inserted into the respective reservoirs 3 or electrodes provided to the respective reservoirs 3 in advance, so that the sample travels uniformly in the loading channel 4 in an electrophoretic manner without going into the separating channel 5 from a crossing point 6. Accordingly, the sample is guided to the crossing point 6 of the loading channel 4 and the separating channel 5.
Next, the applied voltage is switched to the separating channel 5. Also, the voltage is applied to the loading channel 4 so that the sample is moved in a reverse direction from the crossing point 6. Accordingly, only the sample at the crossing point 6 is introduced into the separating channel 5 to carry out the electrophoretic separation. A detector is disposed at a suitable position of the separating channel 5 for detecting a separated component.
As described above, only the sample present at the crossing point 6 is introduced into and separated at the separating channel 5. An actual volume of the sample for the analysis is only from several pico-liters (hereinafter referred to “pl”) to several hundreds pl. However, it is necessary to inject several μl of the sample into the microfluidic device. Therefore, a large quantity of the sample is not used for the analysis. The microfluidic device described above is sometimes used for analyzing a very expensive sample. Therefore, if the analysis requires only a small quantity of a sample, it is possible to reduce the running cost. Thus, there has been a strong demand for reducing a quantity of a sample.
In view of the above problems, the present invention has been made, and an object of the invention is to provide a microfluidic device having a structure in which it is possible to prevent an unnecessary sample from being injected.
Further objects and advantages of the invention will be apparent from the following description of the invention.